1. Field of the Invention
The present invention relates generally to the fields of tuberculosis treatment, enzymology and drug screening. More specifically, the present invention relates to substituted aryl- and heteroaryldiketo acids and derivatives thereof as therapeutics in the treatment of tuberculosis, particularly persistent tuberculosis, and their design.
2. Description of the Related Art
Despite the discovery of streptomycin and several other outstanding anti-tubercular drugs in the 1950's, Mycobacterium tuberculosis (Mtb) continues to cause more deaths worldwide than any other infectious disease. Approximately one-third of the world's population is infected with Mtb, and 8 to 10 million new cases of TB are reported each year. Tuberculosis is estimated to kill about 2 to 3 million people each year, and these deaths are primarily associated with the poor of developing countries.
Mtb is an airborne threat that can be spread by coughs, sneezes, and even droplets from normal speech. In areas of high population density, TB can be spread very rapidly. TB infection rates are on the rise due in part to the HIV epidemic, as there is a synergistic relationship between HIV and TB, where both diseases progress much faster in the co-infected individual. TB has become the leading cause of death for people with HIV (13% of deaths worldwide).
While many new drugs discovered over the following 20 years showed tremendous promise for TB chemotherapy, reports of drug resistance invariably followed shortly after their introduction to the market. Chemotherapy for TB now uses a treatment of multiple antibiotics, e.g., isoniazid, pyrazinamide, ethambutol, and rifampin, that lasts between 6 to 12 months for drug sensitive infection. One major obstacle in combating Mtb during the course of the treatment is the bacteria's ability to survive for extended periods of time in the body in a non-replicative state, referred to as the persistent phase of infection. During this time, the bacteria are characterized as being recalcitrant to treatment by conventional anti-TB drugs and are able to evade the host immune response. This phenotypic resistance dictates the requirement for prolonged drug treatments and oftentimes results in patient non-compliance.
Another threat to the global control of TB is the asymptomatic latent infection. Asymptomatic infections can be activated unpredictably, especially if the patient becomes immunocompromised. The degree of similarity in the metabolic state between bacilli in the persistent and latent phases is not known. However, it is this inherent aspect of the bacteria—its ability to survive in the body for extended periods of time-which reduces the efficacy of current treatments. Drug-resistant strains to at least one TB drug have been found in every country in the world. Many factors contribute to the rise of drug resistance, including low compliance with therapy regimens, poor quality or counterfeit drugs, improper use of drugs, and inadequate health care systems.
In mycobacteria, persistence appears to involve a switch of metabolism to the glyoxylate shunt, which facilitates a metabolic shift in the carbon source to the acetyl CoA generated by the β-oxidation of fatty acids. The glyoxylate shunt enzymes isocitrate lyase (ICL) and malate synthase have been implicated as virulence or persistence factors in several different pathogens. Two genes of the glyoxylate shunt, icl1 and mls1, encoding isocitrate lyase and malate synthase, respectively, were induced upon phagocytosis of Candida albicans by macrophages. A mutant of C. albicans lacking icl1 was less virulent in mice than wild-type. The glyoxylate pathway has also been implicated in the pathogenesis of Brucella abortus and Rhodococcus equi, as well as in the virulence of the plant pathogens Rhodococcus fascians and Stagonospora nodorum. 
For Mtb, it has been reported that genes encoding both of the enzymes are up-regulated in response to phagocytosis. Two genes encode for ICL enzymes in Mtb: icl1 and icl2. While the primary substrate for icl1 is isocitrate, it also possesses a methyl-isocitrate lyase activity, part of a distinct pathway, though some evidence exists for a partial overlap in functionality. An icl1 null strain of Mtb is able to establish an acute infection in mice, but is not able to sustain the chronic, persistent infection seen in mice infected with wild-type Mtb. Moreover, bacteria lacking both icl1 and icl2 are unable to grow on fatty acids or in macrophages, and are rapidly cleared from the lungs of infected mice. It has been reported that antibodies to malate synthase were found in 90% of patients during incipient subclinical tuberculosis. As neither of the genes encoding enzymes in the glyoxylate shunt are found in mammals, both MS and ICL have become targets for the design of drugs.
The anaplerotic maintenance of the tricarboxylic acid (TCA) cycle is afforded by the glyoxylate shunt during conditions where the generation of pyruvate from glycolysis is reduced and beta-oxidation of fatty acids provides the major source of carbon. This metabolic perturbation is vital to the survival of the bacteria. Beta-Oxidation of fatty acids results in increased levels of acetyl CoA, which gets incorporated into isocitrate. ICL converts isocitrate to glyoxylate and succinate. The next enzyme in the glyoxylate cycle, malate synthase, condenses glyoxylate and acetyl CoA to produce malate. An advantage of the glyoxylate cycle, during conditions where beta-oxidation is high, is the bypass of the OO2— generating steps of the TCA cycle. When working in concert with the TCA cycle, the glyoxylate shunt will replenish the concentration of two intermediates, succinate and malate, which will have the overall effect of maintaining oxaloacetate levels (as the entry point for acetyl CoA in the TCA cycle).
In the Mtb genome, a single malate synthase gene (called glcB or mls1) encodes a malate synthase isoform (malate synthase G, Rv1837c). Malate synthase is an 80 kDa (741 amino acids) monomeric protein, homologous to malate synthase (AceB) of the gram-positive bacterium Corynebacterium glutamicum (Reinscheid et al. 1994) and the gram-negative Escherichia coli, with ˜60% sequence identity. Functional properties of malate synthase from Mtb have been studied as well. The specific activity of the purified enzyme was 6 μmol/min/mg protein. The Km of the recombinant protein was determined to be 57 μM for glyoxylate and 30 μM for acetyl CoA. In the absence of divalent cations only negligible activity was measured for the purified enzyme. Mg+2 at 5 mM was found to be the most effective cation. Mn+2 was able to replace Mg+2, yielding 40% of the activity obtained with Mg+2. Co+2, Fe+2, Ca+2, Ba+2, Ni+2, Cd+2, Zn+2, Cu+2, and Hg+2 were unable to support significant activity. The optimal pH for malate synthase activity was found to be 7.5. (Smith et al. 2003).
Also, the inhibition of Mtb malate synthase activity by several compounds, known to be effective against malate synthases, was examined. Oxalate, phosphoenolpyruvate, and bromopyruvate were the most potent inhibitors with inhibition constants of 400, 200, and 60 μM, respectively. Malate was shown to inhibit the activity to ˜50% at 1 mM concentration. 3-Phosphoglycerate, 6-phosphogluconate, fructose-1,6-bisphosphate, and malonic acid had no inhibitory effect at relevant concentrations. Glycolate showed inhibition only at fairly high concentrations (Ki of 900 μM), which is in contrast to other malate synthases such as the enzyme from C. glutamicum, which has a Ki of 440 μM.
Thus, there is a recognized need in the art for tuberculosis drugs effective to treat persistent strains of Mycobacterium tuberculosis. Specifically, the prior art is deficient in novel substituted aryl- and heteroaryldiketo acids and derivatives thereof in the treatment of tuberculosis. The present invention fulfills this long-standing need and desire in the art.